CN111334467A - Photomagnetic bimodal indicator cell, preparation method thereof, detection method thereof and application thereof in-vivo tracing - Google Patents

Abstract

The invention provides a method for preparing a photo-magnetic dual-modal indicator cell, which comprises: suspending red blood cells in a cell isotonic solution, adding fluorescent dyes and a nuclear magnetic contrast agent to obtain a dyeing mixture; and incubating the dyeing mixture, and then using The cells in the staining mixture are washed with isotonic solution of cells, and finally the cells are harvested to obtain the optical and magnetic dual-modality indicator cells. The opto-magnetic dual-modality indicator cell prepared by the preparation method uses the cell as the opto-magnetic dual-modality tracer, which can be used for fluorescence and nuclear magnetic resonance dual-modal imaging, and can be used to reflect biological physiology from the cellular level to the overall level. situation.

Description

A kind of opto-magnetic dual-mode indicator cell, its preparation method, its detection method and its in vivo Applications within Tracer

technical field

The invention belongs to the field of biological imaging, and in particular relates to an opto-magnetic dual-mode indicator cell, a preparation method thereof, a detection method thereof and its application in in vivo tracing.

Background technique

In recent years, the development of bioimaging technology enables the imaging object to be the body, tissue, cell, and even the molecular level, which can reflect the changes at the molecular level in the living state, and conduct qualitative and quantitative analysis of its biological behavior, which promotes drug research. and clinical development.

The opto-magnetic dual-modality imaging technology, by combining fluorescence and MRI imaging probes, enables detection by two imaging techniques, overcoming the inherent limitations of a single imaging technique. MRI is one of the most mature imaging technologies at present. It can provide high-resolution tissue information and three-dimensional structural imaging at the deep sub-millimeter level of the body, and has been widely used in scientific research and clinical practice. However, due to the low imaging resolution and sensitivity, it is difficult to reach the micron level, and clinical application is greatly limited. However, optical microscopy imaging technology has quite high sensitivity, and the detection limit can reach 0.1-0.2 microns. Therefore, the combination of MRI and optical imaging technology can not only provide high-resolution structural histological information, but also realize deep structure. It can achieve the perfect unity of functional imaging and structural tissue imaging.

Optical and magnetic imaging require the help of photomagnetic molecular probes to enhance the signal. At present, the design of common opto-magnetic dual-modal probes is mainly based on two modes: the first is that fluorescent dye molecules directly or indirectly chelate nuclear magnetic signal elements. The second method is to use nanotechnology to construct functionalized nanobimodal systems, which are composed of fluorescent dyes or magnetic particles modified on nanomaterials. However, the opto-magnetic dual-modality probes obtained by the above two methods are in solution state, and it is difficult to reflect the physiological conditions of organisms at the cellular level. out of the blood vessel wall and distributed into the surrounding tissue of the blood vessel wall.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for preparing a photo-magnetic dual-mode indicator cell, and the prepared photo-magnetic dual-mode indicator cell can be used for fluorescence imaging and nuclear magnetic resonance imaging.

Another object of the present invention is to provide an opto-magnetic dual-mode indicator cell, which can be used for fluorescence imaging and nuclear magnetic resonance imaging, and can be used to observe the morphology of red blood cells.

Yet another object of the present invention is to provide a method for detecting the opto-magnetic dual-mode indicator cells, which can perform fluorescence imaging and nuclear magnetic resonance imaging on the opto-magnetic dual-mode indicator cells.

Another object of the present invention is to provide an application of opto-magnetic dual-mode indicator cells in in vivo tracking, which can realize in-vivo tracking of opto-magnetic dual-mode indicator cells under fluorescence imaging and nuclear magnetic resonance imaging.

The invention provides a method for preparing a photo-magnetic dual-modal indicator cell, which comprises: suspending red blood cells in a cell isotonic solution, adding fluorescent dyes and a nuclear magnetic contrast agent to obtain a dyeing mixture; and incubating the dyeing mixture, and then using The cells in the staining mixture are washed with isotonic solution of cells, and finally the cells are harvested to obtain the optical and magnetic dual-modality indicator cells.

The optical and magnetic dual-mode indicator cells prepared by the preparation method can be used for fluorescence and nuclear magnetic resonance dual-mode imaging, and can be used to reflect the physiological conditions of organisms from the cellular level to the overall level.

In another exemplary embodiment of the method for preparing a photo-magnetic dual-modal indicator cell, the fluorescent dye is selected from one of fluorescein CH dipotassium salt, fluorescein isothiocyanate, rhodamine B and Alexa Fluor 594 or a combination of more than one; the MRI contrast agent is gadopentetate meglumine. Thereby, better fluorescence imaging and nuclear magnetic resonance imaging effects can be achieved.

In yet another exemplary embodiment of the method for preparing a photo-magnetic dual-modal indicator cell, the final concentration of fluorescein CH dipotassium salt is not less than 0.001 mM, and the final concentration of fluorescein isothiocyanate is not less than 0.001 mM , the final concentration of rhodamine B is not less than 0.005mM, the final concentration of Alexa Fluor 594 is not less than 0.005mM; the final concentration of gadopentetate meglumine is not less than 0.1mM. Thereby, better fluorescence imaging and nuclear magnetic resonance imaging effects can be achieved. More preferably, the final concentration of fluorescein CH dipotassium salt is not less than 0.1 mM, the final concentration of fluorescein isothiocyanate is not less than 0.1 mM, the final concentration of Rhodamine B is not less than 5 μM, and the final concentration of Alexa Fluor594 Not less than 0.5mM; the final concentration of gadopentetate meglumine is not less than 7.5mM, in order to further improve the effect of fluorescence imaging and nuclear magnetic resonance imaging.

In yet another exemplary embodiment of the method for preparing a photo-magnetic dual-modal indicator cell, the red blood cells are immobilized red blood cells, and the preparation method is: placing the live red blood cells in a cell isotonic solution containing 4% paraformaldehyde, Incubate at 20 to 25°C for no less than 30 minutes. Thereby, better fluorescence imaging and nuclear magnetic resonance imaging effects can be achieved.

In yet another exemplary embodiment of the method for preparing the optical-magnetic dual-modal indicator cells, the method for obtaining live red blood cells is: anticoagulation of blood with an anticoagulant, then centrifugation to remove serum, and cell isotonic solution to wash the blood in the blood The cells were harvested by centrifugation to obtain live red blood cells. Thereby, better fluorescence imaging and nuclear magnetic resonance imaging effects can be achieved.

In yet another exemplary embodiment of the method for preparing the opto-magnetic dual-modal indicator cells, the incubation of the staining mixture is performed at 20 to 25° C. for 0.5 to 72 hours, preferably 6 to 72 hours.

In yet another exemplary embodiment of the method for preparing the optical and magnetic dual-modality indicator cells, the cell isotonic solution is physiological saline. Thereby, interference can be reduced, so as to achieve better fluorescence imaging and magnetic resonance imaging results.

In yet another exemplary embodiment of the method for preparing the opto-magnetic dual-modality indicator cells, the method for harvesting the cells is centrifugation.

The present invention also provides an opto-magnetic dual-mode indicator cell, which is prepared by the above preparation method. The optical and magnetic dual-modality indicator cells can be used for fluorescence and nuclear magnetic resonance dual-modality imaging, and can be used to reflect the physiological conditions of organisms from the cellular level to the overall level.

The present invention also provides a method for detecting the opto-magnetic dual-mode indicator cells, which comprises: resuspending the opto-magnetic dual-mode indicator cells in a cell isotonic solution, then placing them in a laser scanning confocal microscope for detection, and passing through a fluorescence channel and light microscope imaging; in which the parameters of the fluorescence channel are determined according to the excitation and emission wavelengths of the fluorescent dyes; and the opto-magnetic dual-mode indicator cells are placed in an EP tube and diluted with cell isotonic solution until the number of cells is 10 ⁴ to 10 ⁸ cells /mL, and then the EP tube was placed in a magnetic resonance imaging system (MRI) for imaging. The detection method can perform fluorescence imaging and nuclear magnetic resonance imaging on the optical-magnetic dual-mode indicator cells. The parameters of MRI imaging are preferably: use wrist coil and use T1 3D MP-RAGE sequence to scan, and imaging parameters are: echo time (TE)=3.7ms, repetition time (TR)=1500ms, reversal angle (FA) =90, inversion time (TI)=900ms, slice thickness (SL)=1mm, field of view (FOV)=267mm, matrix 512×96, resolution=0.5mm×0.5mm×0.5mm.

The present invention also provides an application of the above opto-magnetic dual-mode indicator cells in in vivo tracking, which includes: introducing the opto-magnetic dual-mode indicator cells into an animal; placing the animal in a magnetic resonance imaging system for MRI imaging ; and the animals, the tissue sections of the animals or the cells of the animals are placed in a laser scanning confocal microscope for detection, wherein the parameters of the fluorescence channel are determined according to the excitation and emission wavelengths of the fluorescent dyes. The application can realize the in vivo tracking of optical and magnetic dual-modal indicator cells under fluorescence imaging and nuclear magnetic resonance imaging, so as to reflect the physiological status of organisms at the cellular level.

In another exemplary embodiment of the application of opto-magnetic dual-modality indicator cells in in vivo tracking, the parameters of MRI imaging are: using a wrist coil and using T1 3D MP-RAGE sequence to scan, and the imaging parameters are: back Wave time (TE)=3.7ms, repetition time (TR)=1500ms, reversal angle (FA)=90, reversal time (TI)=900ms, slice thickness (SL)=1mm, field of view (FOV)=267mm, Matrix 512×96, resolution=0.5mm×0.5mm×0.5mm.

In another exemplary embodiment of the application of the opto-magnetic dual-modality indicator cells in in vivo tracking, the opto-magnetic dual-modality indicator cells are introduced into the cistern magnum of an animal, and the part of the tissue section is the brain tissue of the animal. The application can use fluorescence imaging and magnetic resonance imaging to reflect the physiological state of the animal brain from the cellular level.

Description of drawings

The following drawings merely illustrate and explain the present invention schematically, and do not limit the scope of the present invention:

Fig. 1 is the fluorescence image (A), light microscope image (B) and fluorescence light microscope stack image (C) of 488nm excitation light of the photo-magnetic dual-mode indicator cell corresponding to No. 6;

Fig. 2 is the 488nm excitation light fluorescence image (A), light microscope image (B) and fluorescence light microscope stack image (C) of the opto-magnetic dual-mode indicator cell corresponding to No. 13;

Fig. 3 is the fluorescence image of 488nm excitation light (A), the fluorescence image of 633nm excitation light (B), the light microscope image (C) and the fluorescence light microscope overlay (D) of the opto-magnetic dual-modal indicator cell corresponding to No. 19;

Figure 4 is an MRI image of the subarachnoid region after spraying;

Figure 5 is an image obtained by laser scanning confocal microscopy of the subarachnoid space after spraying.

Detailed ways

In order to have a clearer understanding of the technical features, purposes and effects of the invention, the specific implementations of the invention will now be described with reference to the following examples.

Reagents and instruments used in the following examples:

Laser scanning confocal microscope (LSCM) is Leica TCS SP8;

Magnetic resonance imager (3.0T, Trio) from Siemens, Germany;

Stereotaxic apparatus for small animal experiments was purchased from Stoelting, USA;

Rats were purchased from the Animal Department of Peking University School of Medicine;

The fluorescent yellow CH dipotassium salt fluorescent probe was purchased from Sigma-Aldrich Co.LLC.;

Fluorescein isothiocyanate was purchased from life technology in the United States;

Rhodamine B was purchased from Sarn Chemical Technology (Shanghai) Co., Ltd.;

Alexa fluor 594 was purchased from American life technology;

Brain slice molds were purchased from Shenzhen Reward Life Technology Co., Ltd.;

The high-speed centrifuge was purchased from Beckman, USA;

Physiological saline (500ml) was purchased from Beijing Soleibao Technology Co., Ltd.;

4% tissue cell fixative (4% paraformaldehyde) was purchased from Beijing Soleibao Technology Co., Ltd.;

Confocal microscope glass bottom petri dishes were purchased from Zhongke Maichen (Beijing) Trading Co., Ltd.;

Micro-syringes were purchased from Hamilton Company of the United States;

Gadopentetate meglumine injection was purchased from Beijing Beilu Pharmaceutical Co., Ltd.

Example 1: Preparation and detection of opto-magnetic dual-modal indicator cells.

1. Steps for preparing opto-magnetic dual-mode indicator cells:

1.1. Collect blood from the heart of SD rats, anticoagulate the blood with EDTA, then remove the serum by centrifugation and wash the cells in the blood with normal saline 3-4 times, and finally centrifuge at 2000r/min for 10min to harvest the cells to obtain live red blood cells (due to red blood cells and white blood cells). The proportions of leukocytes are very different, and generally white blood cells are mixed in them, which can be ignored);

1.2. Take live red blood cells and place them in physiological saline containing 4% paraformaldehyde (ie, 4% tissue cell fixative), and incubate at 20 to 25°C for 30 minutes (this time can be extended according to the actual situation, as long as The cells do not rupture) to obtain immobilized red blood cells, wherein the volume ratio of the immobilized red blood cells to the physiological saline containing 4% paraformaldehyde is 1:5;

1.3. Resuspend the immobilized red blood cells in physiological saline, and add fluorescent dyes and MRI contrast agents to obtain a dyeing mixture. The types of fluorescent dyes and NMR contrast agents and the final concentrations in the dyeing mixture are shown in Table 1;

1.4. Incubate the staining mixture at 20 to 25° C. for 48 hours, then wash the cells in the staining mixture with physiological saline, and finally harvest the cells by centrifugation to obtain optical-magnetic dual-modality indicator cells.

2. The steps of detecting the opto-magnetic dual-mode indicator cells:

2.1. The opto-magnetic dual-mode indicator cells were resuspended in physiological saline, dropped into a confocal glass dish, and then placed in a laser scanning confocal microscope for detection. The fluorescence channel and light microscope imaging were used to measure the average single cell fluorescence intensity. Table 1 below; the parameters of the fluorescence channel are determined according to the corresponding excitation and emission wavelengths of the disclosed fluorescent dyes, which are not repeated here; the opto-magnetic dual-mode indicator cells used are the opto-magnetic dual-mode just prepared indicator cells;

2.2. Put the opto-magnetic dual-mode indicator cells in an EP tube, dilute with normal saline to a cell number of 10 ⁶ cells/mL (the cell concentration can be adjusted to 10 ⁴ to 10 ⁸ cells/mL according to the actual situation), and then The EP tube was placed in a 3.0T magnetic resonance imaging system for MRI imaging, and a wrist coil was used for scanning with a T1 3D MP-RAGE sequence. The imaging parameters were: echo time (TE)=3.7ms, repetition time (TR)=1500ms , inversion angle (FA)=90, inversion time (TI)=900ms, layer thickness (SL)=1mm, field of view (FOV)=267mm, matrix 512×96, resolution=0.5mm×0.5mm×0.5mm . The measured NMR signal intensities are shown in Table 1 below.

Table 1:

Figure 1 shows the 488nm excitation light fluorescence image (A), light microscope image (B) and fluorescence light microscope overlay (C) of the opto-magnetic dual-mode indicator cell corresponding to No. 6 in Table 1 above.

Figure 2 shows the 488nm excitation light fluorescence image (A), light microscope image (B) and fluorescence light microscope overlay (C) of the opto-magnetic dual-modal indicator cell corresponding to No. 13 in Table 1 above.

Fig. 3 shows the fluorescence image of 488nm excitation light (A), 633nm excitation light fluorescence image (B), light microscope image (C) and fluorescence light microscope overlay ( D).

According to the above Table 1 and Figures 1-3, it can be seen that the optical and magnetic dual-modality indicator cells prepared in the above No. 1-19 can be used for fluorescence-MRI dual-modality imaging.

Physiological saline used in this example can be replaced with other isotonic fluids for cells.

Although only red blood cells are used in the above embodiments, it is not limited thereto, and other types of cells may also be used in other exemplary embodiments.

Example 2 (Incubation time).

The incubation time in step 1.4 in Example 1 was set to 6 hours, 48 hours and 72 hours, respectively. The types of fluorescent dyes and nuclear magnetic contrast agents and their final concentrations in the staining mixture were shown in No. 13 in Table 1, and other parameters remained unchanged. . The average single cell fluorescence intensity and nuclear magnetic signal intensity of the prepared opto-magnetic dual-modal indicator cells are shown in Table 2 below.

Table 2:

single cell fluorescence intensity NMR signal strength Incubation time: 6 hours 178.212±7.62 489.03±9.91 Incubation time: 48 hours 252.316±3.54 877.34±12.54 Incubation time: 72 hours 252.623±2.32 834.45±13.62

As shown in Table 2, when the incubation time was 6 hours, the fluorescence intensity and NMR signal intensity of a single cell reached high contrast, and fluorescence imaging and NMR imaging could be achieved. When the incubation time reached 48 hours, the photomagnetic cell spheroids reached the optimal single cell fluorescence intensity and NMR signal intensity. Continue to increase the incubation time to 72 hours, the single cell fluorescence intensity and NMR signal intensity reach a plateau and no longer increase.

Example 3 (stability).

The photo-magnetic dual-modal indicator cells corresponding to No. 13 in Table 1 of Example 1 were placed in the dark at room temperature (20-25° C.) for 1 day and 7 days respectively after preparation, and then the average single cell fluorescence intensity and NMR signal strength, the detection results are shown in Table 3 below.

table 3:

single cell fluorescence intensity NMR signal strength Detection time: 1 day 252.316±3.54 877.34±12.54 Detection time: 7 days 220.293±2.37 690.21±11.67

.

It can be seen from the above Table 3 that the optical and magnetic dual-modality indicates that the fluorescence signal and the nuclear magnetic signal of the cells can be kept basically stable within 7 days of storage.

Example 4: Application of opto-magnetic dual-modal indicator cells in in vivo tracking.

step:

1. 250g rats were anesthetized, skin prepared, and labeled; the rats were fixed with a brain stereotaxic instrument, and 50 μL of the opto-magnetic dual-modal indicator cells corresponding to No. 13 in Table 1 of Example 1 were injected into the cisterna magna; Then, the rats were placed in a 3.0T magnetic resonance imaging system for MRI imaging, and the wrist coil was used for scanning with T1 3D MP-RAGE sequence. The imaging parameters were: echo time (TE)=3.7ms, repetition time (TR )=1500ms, inversion angle (FA)=90, inversion time (TI)=900ms, slice thickness (SL)=1mm, field of view (FOV)=267mm, matrix 512×96, resolution=0.5mm×0.5mm ×0.5mm; the results are shown in Figure 4, where row A is the sagittal MRI image at 0, 15, 30, and 60 min, row B is the horizontal axial MRI image at 0, 15, 30, and 60 min, and row C is coronal MRI images at positions 0, 15, 30, and 60 minutes; it can be seen that the subarachnoid space area after spraying has stronger positive signals than the pre-scan group (0 minutes);

2. Anesthetize the rat, perfuse the heart, take the brain, and place it in 4% paraformaldehyde for 2 hours; place the fixed rat brain in the rat brain mold and slice coronally, each slice is about 2 mm thick, to obtain The slices were examined by a laser scanning confocal microscope, in which the parameters of the fluorescence channel were determined according to the excitation and emission wavelengths of the fluorochromes. The detection results are shown in Figure 5, in which A is the fluorescence image, B is the light microscope image, C is the reflected light image, D is the fluorescent light microscope stack image, and E is the partial enlarged image of A; the fluorescence distribution can be seen from the figure. in the subarachnoid region of the rat brain.

It can be seen that the opto-magnetic dual-modal indicator cells can be tracked in vivo under two imaging techniques of MRI imaging and fluorescence imaging, thereby reflecting the physiological conditions of organisms at the cellular level. The opto-magnetic dual-mode indicator cell has the dual characteristics of fluorescent dye and nuclear magnetic contrast agent at the same time, and the two detection technologies can be combined. After it is injected into the animal, it can be used to determine the physiological state of the animal according to its distribution characteristics in the body.

In addition, the opto-magnetic dual-mode indicator cells located in animal blood vessels can dynamically monitor the blood flow velocity of blood vessels in real time under the laser confocal microscope and nuclear magnetic imager, and can clearly observe the flow process of opto-magnetic cell spheres in the blood vessels. Therefore, it can be used for vascular tissue imaging. And because it exists in the form of cells, there is no problem of permeating the blood vessel wall, so that the physiological condition of the organism can be more accurately reflected from the cellular level.

As used herein, "exemplary" means "serving as an example, instance, or illustration", and any implementation described herein as "exemplary" should not be construed as a more preferred or more advantageous technical solution.

In this paper, "equal", "same" and the like are not limitations in strict mathematical and/or geometric senses, but also include errors that can be understood by those skilled in the art and allowed in production or use. Unless otherwise indicated, numerical ranges herein include not only the entire range between its two endpoints, but also several subranges subsumed therein.

It should be understood that although this specification is described according to various embodiments, not each embodiment only includes an independent technical solution, and this description in the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole , the technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.

The series of detailed descriptions listed above are only specific descriptions for the feasible embodiments of the present invention, and they are not used to limit the protection scope of the present invention. Changes, such as combination, division or repetition of features, should be included within the scope of protection of the present invention.

Claims (9)

1. A preparation method of a photomagnetic bimodal indicator cell is characterized by comprising the following steps:

suspending erythrocytes in cell isotonic solution, and adding a fluorescent dye and a nuclear magnetic contrast agent to obtain a staining mixture; and

and incubating the staining mixture, washing the cells in the staining mixture by using a cell isotonic solution, and finally harvesting the cells to obtain the photomagnetic bimodal indicator cell.

2. The method for preparing a magneto-optical bimodal indicator cell according to claim 1, wherein the fluorescent dye is selected from a combination of one or more of fluorite CH dipotassium salt, fluorescein isothiocyanate, rhodamine B and Alexa Fluor 594; the nuclear magnetic contrast agent is gadopentetate dimeglumine.

3. The method for preparing a magneto-optical bimodal indicator cell as claimed in claim 2, wherein the final concentration of fluorite CH dipotassium salt is not less than 0.001mM, the final concentration of fluorescein isothiocyanate is not less than 0.001mM, the final concentration of rhodamine B is not less than 0.005mM, and the final concentration of Alexa Fluor594 is not less than 0.005 mM; the final concentration of the gadopentetate dimeglumine is not less than 0.1 mM.

4. The method of claim 1, wherein the red blood cells are immobilized red blood cells, and the method comprises incubating live red blood cells in an isotonic solution of cells containing 4% paraformaldehyde at 20-25 ℃ for no less than 30 minutes.

5. The method for preparing magneto-optical bimodal indicator cell according to claim 4, wherein the method for obtaining the living red blood cell is: anticoagulating blood with anticoagulant, centrifuging to remove serum, washing cells in the blood with cell isotonic solution, and centrifuging to obtain the living red blood cells.

6. A magneto-optical bimodal indicator cell, characterized in that it is obtained by the method of any one of claims 1 to 5.

7. The method for detecting the magneto-optical bimodal indicator cell according to claim 6, comprising:

resuspending the photomagnetic bimodal indicator cells in cell isotonic solution, then placing the cell isotonic solution in a laser scanning confocal microscope for detection, and imaging through a fluorescence channel and an optical lens; wherein parameters of the fluorescent channel are determined from excitation and emission wavelengths of the fluorescent dye; and

placing the photomagnetic bimodal indicator cell in an EP tube, and diluting the indicator cell with cell isotonic solution until the cell number is 10⁴To 10⁸cell/mL, and then placing the EP tube in a magnetic resonance imaging system for imaging.

8. Use of the magneto-optical bimodal indicator cell according to claim 6 for in vivo tracking, comprising:

introducing the photomagnetic bimodal indicator cell into an animal;

placing the animal in a magnetic resonance imaging system for MRI imaging; and

placing said animal, tissue section of said animal, or cells of said animal in a confocal laser scanning microscope for detection, wherein parameters of said fluorescent channel are determined based on excitation and emission wavelengths of said fluorescent dye.

9. The use of the magneto-optical bimodal indicator cell in vivo tracking as claimed in claim 8, wherein the magneto-optical bimodal indicator cell is introduced into the occipital cisterna of an animal, and the tissue section is the brain tissue of the animal.

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